Photostable dyes, which exhibit a substantial to highly pronounced absorption in the whole visible spectral range, a moderate to high quantum fluorescence yield, and a large Stokes shift in the near infrared (NIR) spectral range, and exhibit only a weak solvatochromic effect and have no net charge (that is, carry a neutral charge or a zwitterionic charge), are suitable as marker and reference materials in chemical, physical, or biological applications that are based on the measurement of fluorescence signals.
The dyes that are commercially available today are characterized either by
(i) narrow and pronounced absorption bands that are in the NIR which exclude an excitation and use in the visible range, have narrow fluorescence bands and small Stokes shifts, and have only low to moderate quantum fluorescence yields and low photostability (for example, cyanine dyes),
(ii) by wide absorption bands, but only narrow fluorescence bands that exhibit a small Stokes shift, with low quantum fluorescence yields that deliver only weak signals (for example, porphyrins),
(iii) by wide absorption bands and fluorescence bands that exhibit a strong Stokes shift, but only low quantum fluorescence yields and a strong solvatochromic effect, i.e., a high sensitivity to changes in their immediate environment (for example, styryl or oxazine dyes),
(iv) by narrow and pronounced absorption bands that are in the NIR which exclude an excitation and use in the visible range, and that have narrow fluorescence bands and small Stokes shifts, but have high quantum fluorescence yields and high photostability (for example, terrylenimide dyes),
(v) by good photostability and wide absorption and emission bands, but only low quantum fluorescence yields (for example, quaterrylenebis(dicarboximide) dyes), or
(vi) by narrow and pronounced absorption bands that are in the NIR which exclude an excitation and use in the visible range, and that have small Stokes shift and a sensitivity to nucleophilic species (for example, squaraine dyes).
In addition, most of the currently commercially available NIR dyes, with the exception of porphyrins, squaraines, and perylene derivatives, carry a net charge (usually a positive charge), a state that limits their application to polar solvents, materials, and environments.
The results are the following drawbacks:
(i) narrow absorption bands=no possibility of wide-band excitation and use,
(ii) small Stokes shifts=only inadequate separation from excitation light/scattering and fluorescence signal possible,
(iii) low quantum fluorescence yields=weak fluorescence signals,
(iv) low photostability=inadequate service life of the dye or component, and
(v) pronounced solvatochromism=undesired signal fluctuation in the event that the environmental parameters change.
The object of the invention is to provide electronically neutral NIR-emitting dyes as potent fluorophores, markers, or reference materials for a wide variety of NIR fluorometric applications, which exhibit better properties, that is, wide-band excitation possibilities, intensive NIR fluorescence, large Stokes shift, high photostability, low solvatochromic effect, and an insensitivity to environmental influences.
In this context, the class of difluoroboryl complexes of dipyrrin (also 4,4-difluoro-4-boro-3a,4a-diaza-s-indacene) is especially important because of the obvious fluorescence of this class of compounds. Representatives of this class of compounds are marketed under the tradename BODIPY (from the English for BOron DIPYrrin) as fluorescence markers for application in the field of molecular biology. In addition to its original use as a biolabel, the dye is also used as a cation sensor, laser dye, and in other fields of the material sciences. The dyes are relatively easily synthetically accessible with a variable substitution pattern and are accessible in preparative quantities. The reason for the multifaceted application and extremely wide spectrum of successful applications of this class of dyes can be attributed to some basic properties, such as high molar extinction coefficient (ε>80,000 M−1 cm−1), high quantum fluorescence yield (φ>0.70), and moderate redox potential. Representatives of this class of dyes can be found, for example, in U.S. Pat. No. 5,248,782; U.S. Pat. No. 6,005,113; E. Deniz et al., Organic Letters (2008), Vol. 10, No. 16, pages 3401-3403; S. Erten-Ela et al., Organic Letters (2008), Vol. 10, No. 15, pages 3299-3302; Q. Zheng et al., Chem. Eur. J. (2008), No. 14, pages 5812-5819; and Ziessel et al., Chem. Eur. J. (2009), No. 15, pages 1359-1369.
The drawback with the BODIPY dye is that its use is typically limited to a wavelength range between 470 and 530 nm. In addition, a Stokes shift ranging from 5 to 15 nm is a limiting factor for many applications. In particular, this property is a drastic disadvantage for use in single molecule spectroscopy and in multiplexing applications.